CN102405279A - Method for improved single cell cloning - Google Patents

Method for improved single cell cloning Download PDF

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CN102405279A
CN102405279A CN2010800143255A CN201080014325A CN102405279A CN 102405279 A CN102405279 A CN 102405279A CN 2010800143255 A CN2010800143255 A CN 2010800143255A CN 201080014325 A CN201080014325 A CN 201080014325A CN 102405279 A CN102405279 A CN 102405279A
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CN102405279B (en
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科利亚·黑格勒
奥拉夫·克吕格尔
阿齐兹·恰伊勒
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Cellca GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0056—Xeno-free medium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0043—Medium free of human- or animal-derived components
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Abstract

The present invention relates to methods for the cultivation of a population of cells in a serum free cell culture medium, wherein the population of cells has a cell concentration of less than 100 cells/ml, wherein a serum free cell culture medium containing recombinant albumin and recombinant transferrin is used.

Description

Improve the method for single cell clone
The present invention relates to culturing cell crowd's in serum-free cell culture medium method, the cell concn of wherein said cell mass has wherein used the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin less than 100 cell/ml.The invention still further relates to the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin and be used for the purposes of culturing cell concentration less than the cell mass of 100 cell/ml.The invention still further relates in the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin cultured cells concentration less than the cell mass of 100 cell/ml.Conventional cell culture medium is supplemented with uncertain additive usually, for example foetal calf serum (FBS).This additive is that carrier provides instability or water insoluble active ingredient, growth factor is provided and has protected cell to avoid physical stress.On the other hand, use serum to have some shortcomings.Serum is the mixture of the material that do not characterize, and it maybe be with batch variation.Yet, be the risk that exogenous factor pollutes, for example mycoplasma, Protein virus and virus pollution from the serum in animal or human source or the main drawback of other fill-in.
In order to overcome the risk that the pathogenicity bo relevant with serum polluted, the past has been developed serum free medium.Serum free medium is supplemented with serum substitute usually, for example growth factor, cytokine, BSA, Regular Insulin and Transferrins,iron complexes.These albumen separate from the animal source usually, so substratum is still existed by the potential risk of pathogen contamination.For example, utilize the bovine serum albumin (BSA), human serum albumin (HSA) or the Transferrins,iron complexes that separate from the animal or human source can be convenient to cell cultures (US6733746).This method still exists in cell culture introduces the exotic disease substance, for example from the risk of HIV, the creutzfeldt-Jacob disease factor (Creutzfeld Jakob agent) or the hepatitis virus of HSA.Pathogenic agent is unfavorable for the application of substratum in the production of animal and human's therapeutical agent.The substratum that therefore protein that only contains the reorganization generation also reduced the risk of pathogenicity bo pollution has been described.Disclose the substratum (WO2008009642) that comprises recombinant albumin and the other composition that is derived from animal and contained recombinant albumin and the substratum of Recombulin (US20060115901).
Usually the conventional additives that in serum free medium, adds is a Transferrins,iron complexes.Transferrins,iron complexes usually separates from the animal or human source, and is added to and thinks in the serum free medium that cell provides iron.Qian, Z.M. and Tang, P.L., 1995, Biochim.Biophys.Acta, 1269:205-214 have summarized the mechanism of mammalian cell picked-up iron.Further publication shows that Transferrins,iron complexes possibly be the growth factor of sustenticular cell propagation.On the other hand; Shown that the TfR deficient cell can be with the speed propagation suitable with wild-type cell, this shows that this receptor does not belong to growth factor receptors family (Chan, R. etc.; 1992, Experimental cell research 202:326-336).Under the situation that lacks TfR, cell can be through the non-dependent mechanism picked-up of non-specific acceptor iron.Usually, the mechanism that cell can absorb iron has three kinds: 1. from Transferrins,iron complexes via the acceptor dependent pathway, 2. from Transferrins,iron complexes via acceptor dependent/non-dependent approach (non-specific), 3. from inorganic molysite, FeSO for example 4(Chan, R. etc., 1992, Experimental cell research 202:326-336).Back one iron picked-up approach is the basis that in substratum, adds many inorganic iron sequestrants, other effect (EP1210410) that it provides iron and ignore Transferrins,iron complexes to cell.Because cell has been developed many any proteinic cell culture mediums that do not contain fully from the non-specific iron picked-up of inorganic molysite.What is interesting is that cell is grown very goodly in not protein-contg substratum, thereby make the substratum of present this area not contain any protein.In a word, disclosed digital proof Transferrins,iron complexes can mediate the picked-up of iron in mammalian cell.Yet because cell can also be from inorganic iron sequestrant picked-up iron, therefore replenishing iron might not need Transferrins,iron complexes.So far, not clear Transferrins,iron complexes has the growth factor-like effect except it as whether going back pair cell iron provider's the effect.
In the process of utilizing the animal cell culture manufacture of therapeutic protein, prove that clone's property of production clone is very important.This expression, the production clone that process is modified in heredity should rise and be derived from single precursor cell, and said precursor cell is only to inoculate the individual cells that a cell is cloned through each hole in petridish.The cellular exposure that makes unicellular state is in fast-changing envrionment conditions, like the deleterious effect of pH variation, temperature variation or accumulative oxidizing medium product.By contrast, being total to cultured cells in the cell mass accepts such as the support propagation of cytokine and the composition of survival from its flanking cell.When omission in the single cell culture process should be supported, many clones can not deal with the cultivation stress of raising.When mono-clonal is seeded to petridish, in the time of for example in 96 orifice plates, the clone can not be from accepting the pungency support on every side.This usually causes necrocytosis or non-propagation behavior.For fear of these difficult problems, in substratum, exist usually under the condition of foetal calf serum (FBS) and carry out single cell clone.Therefore, because the above-mentioned shortcoming of serum, the single cell clone substratum that exploitation does not contain serum is significant.
Developed in the substratum that does not contain serum the method for cultivating Chinese hamster ovary celI with low-down cell density.In addition, said substratum comprises recombinant albumin and Recombulin (US20060115901).Usually in the single cell clone substratum that does not contain serum, add conditioned medium.Conditioned medium comprises the cytokine that is produced by same cell mass and therefore should promote single celled clonal growth (WO2005014799).Yet the low and conditioned medium of the concentration of cytokine also comprises the cytotoxicity metabolite that suppresses growth in the conditioned medium, thereby the growth promoting function of this substratum is restricted.
Another kind method is the common cultivation of actual production clone and parent cell.In order to remove the energy for growth of so-called feeder cell, can make this cell accept radiation.The feeder cell of not growing will discharge stimulating growth clone splitted growth factor (EP1176194, US2005/0059146).The shortcoming of utilizing feeder cell to cultivate is to be difficult to clone from the feeder cell separation of produced.Must prove that feeder cell are not attached to the production clone.
In a word, disclosed digital proof need be used for carrying out at serum free medium the simple novel method of the single cell clone of cell.
Technical problem of the present invention provides the method and the cell culture medium of the shortcoming that overcomes prior art.
Further technical problem of the present invention provides the simpler method of the single cell clone that especially in serum free medium, carries out cell.
Further technical problem of the present invention provides the simpler method that (especially in serum free medium) carries out the cell mass cultivation with low cell concn in substratum.
The present invention has solved the problems referred to above through the instruction that independent claim are provided.
Particularly; The invention provides a kind of in serum-free cell culture medium culturing cell crowd's method; The cell concn of wherein said cell mass is less than 100 cell/ml, and said method comprises the steps: a) in first serum-free cell culture medium with greater than about 100 cell/ml, especially greater than the cell concn culturing cell crowd of 100 cell/ml; B) reduce said cell concn extremely less than about 100 cell/ml; Especially less than 100 cell/ml, and c) in second serum-free cell culture medium, cultivate said cell, wherein said second serum-free cell culture medium comprises recombinant albumin and recombinant transferrin.
Particularly; The invention provides a kind of in serum-free cell culture medium culturing cell crowd's method; The cell concn of wherein said cell mass is less than 100 cell/ml, and said method comprises the steps: a) in first serum-free cell culture medium with greater than about 100 cell/ml, especially greater than the cell concn culturing cell crowd of 100 cell/ml; B) reduce cell concn extremely less than about 100 cell/ml; Especially less than 100 cell/ml, and c) making said cells contacting second serum-free cell culture medium, wherein said second serum-free cell culture medium comprises recombinant albumin and recombinant transferrin.
Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 200 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than about 200 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 500 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than about 1000 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 1000 cells.Of the present invention preferred embodiment in, in step b), said cell concn is reduced to less than 50 cell/ml.Of the present invention preferred embodiment in, in step b), said cell concn is reduced to less than 10 cell/ml.Of the present invention preferred embodiment in, in step b), cell concn is reduced to 1 cell/ml.
Of the present invention preferred embodiment in, in step b), cell mass is reduced to 1 cell.Therefore, of the present invention preferred embodiment in, said cell mass comprises 1 cell when step c) begins.
Of the present invention preferred embodiment in, in step b), said cell mass is reduced to 1 cell/petridish.Therefore, of the present invention preferred embodiment in, said cell mass comprises 1 cell/petridish when step c) begins.
Of the present invention preferred embodiment in, in step b), said cell mass is reduced to 1 cell/culture hole.Therefore, of the present invention preferred embodiment in, said cell mass comprises 1 cell/culture hole when step c) begins.Those skilled in the art know suitable culturing cell, especially culturing cell crowd's several petridish and culture hole.Those skilled in the art also know several petridish and the culture hole of suitable culturing cell concentration less than the cell mass of 100 cell/ml.Those skilled in the art also know and are fit to culturing cell concentration altogether less than the cell mass of 100 cells, especially only by several petridish and the culture hole of a unicellular cell mass of forming.
In addition; The present invention also provides a kind of single celled method of in serum-free cell culture medium, cultivating; Said method comprises the steps: a) that in first serum-free cell culture medium with greater than about 100 cell/ml, especially cultivate greater than the cell concn of 100 cell/ml, cell mass b) is isolated unicellular from said cell mass; And c) cultivation is said unicellular in second serum-free cell culture medium, and wherein said second cell culture medium comprises recombinant albumin and recombinant transferrin
In addition, the present invention also provides a kind of single celled method of in serum-free cell culture medium, cultivating, and said method comprises the steps: a) in first serum-free cell culture medium with greater than about 10 3Individual cell/ml, especially greater than 10 3The cell concn culturing cell crowd of individual cell/ml, b) isolate from said cell mass unicellular, with c) make said unicellularly contact with second serum-free cell culture medium, wherein said second cell culture medium comprises recombinant albumin and recombinant transferrin.
Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 100 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than about 200 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 500 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than about 1000 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 10000 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than about 100000 cells.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 1000000 cells.
Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 100 cell/ml.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than about 200 cell/ml.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 500 cell/ml.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than about 1000 cell/ml.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 10000 cell/ml.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than about 100000 cell/ml.Of the present invention preferred embodiment in, the cell concn in the step a) is greater than 1000000 cell/ml.
Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of about 100 cell/ml the time, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of 100 cell/ml the time, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of about 200 cell/ml the time, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of 200 cell/ml the time, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of about 500 cell/ml the time, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of 500 cell/ml the time, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than about 10 3During the cell concn culturing cell of individual cell/ml, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than 10 3During the cell concn culturing cell of individual cell/ml, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than about 10 4During the cell concn culturing cell of individual cell/ml, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than 10 5During the cell concn culturing cell of individual cell/ml, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than about 10 6During the cell concn culturing cell of individual cell/ml, the cell of said cell mass does not need recombinant transferrin and/or recombinant albumin to be used for growth.
Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of about 100 cell/ml the time, the cell of said cell mass does not need recombinant transferrin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of about 100 cell/ml the time, the cell of said cell mass does not need recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of 100 cell/ml the time, the cell of said cell mass does not need recombinant transferrin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of 100 cell/ml the time, the cell of said cell mass does not need recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of about 1000 cell/ml the time, the cell of said cell mass does not need recombinant transferrin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of about 1000 cell/ml the time, the cell of said cell mass does not need recombinant albumin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of 1000 cell/ml the time, the cell of said cell mass does not need recombinant transferrin to be used for growth.Of the present invention preferred embodiment in, when with greater than the cell concn culturing cell of 1000 cell/ml the time, the cell of said cell mass does not need recombinant albumin to be used for growth.
In preferred implementation of the present invention, the cell of said cell mass is the cell that can in serum free medium, grow.Of the present invention preferred embodiment in, the cell of said cell mass is to adapt to the cell in serum free medium, grow.
Of the present invention preferred embodiment in, the cell of said cell mass is the cell that can in the substratum that does not contain animal component, grow.Of the present invention preferred embodiment in, the cell of said cell mass is to adapt to the cell in the substratum that does not contain animal component, grow.Of the present invention preferred embodiment in, the cell of said cell mass is can be with the cell of in the substratum that does not contain animal component, growing less than the concentration of 100 cell/ml.Of the present invention preferred embodiment in, the cell of said cell mass is the cell that adapts to grow in the substratum that does not contain animal component less than the concentration of 100 cell/ml.
Of the present invention preferred embodiment in, said method comprises other step before the step a), and the cell adapted of said cell mass grown in the substratum that does not contain animal component.In optional embodiment of the present invention, the cell adapted of said cell mass grown in the substratum that does not contain animal component.
Can also pass through, for example use business-like cell and the cell that provides adaptation in the substratum that does not contain animal component, to grow, in step a), to use.
Make cell adapted appropriate method of in the substratum that does not contain animal component, growing, i.e. it is well known in the art that acquisition adapts to the method for the cell of in the substratum that does not contain animal component, growing.
Of the present invention preferred embodiment in, the said substratum that does not contain animal component is not protein-contg substratum.
Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the BSA of concentration less than 0.1% (by weight).Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) the comprises concentration BSA of 0.09% (by weight) at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) the comprises concentration BSA of 0.05% (by weight) at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) the comprises concentration BSA of 0.01% (by weight) at the most.
Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the recombinant albumin of concentration less than 0.1% (by weight).Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) the comprises concentration recombinant albumin of 0.09% (by weight) at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) the comprises concentration recombinant albumin of 0.05% (by weight) at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) the comprises concentration recombinant albumin of 0.01% (by weight) at the most.
Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the Transferrins,iron complexes of concentration less than 5 μ g/ml.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the concentration Transferrins,iron complexes of 4.9 μ g/ml at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the concentration Transferrins,iron complexes of 4 μ g/ml at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the concentration Transferrins,iron complexes of 1 μ g/ml at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the concentration Transferrins,iron complexes of 0.5 μ g/ml at the most.
Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the recombinant transferrin of concentration less than 5 μ g/ml.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the concentration recombinant transferrin of 4.9 μ g/ml at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the concentration recombinant transferrin of 4 μ g/ml at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the concentration recombinant transferrin of 1 μ g/ml at the most.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) comprises the concentration recombinant transferrin of 0.5 μ g/ml at the most.
Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) does not contain recombinant albumin.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) does not contain recombinant transferrin.Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) does not contain recombinant albumin and recombinant transferrin.
Of the present invention preferred embodiment in, said first serum free medium that uses in the step a) does not contain BSA and Transferrins,iron complexes.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that rises greater than 2g/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 2.1g/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 2.5g/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 3g/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 5g/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 10g/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 20g/ at least rises.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that rises greater than 10mg/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 10.1mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 11mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 20mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 50mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 100mg/ at least rises.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant albumin that rises less than 2g/, and the cell culture medium that wherein uses in the step c) comprises the recombinant albumin that rises greater than 2g/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant albumin that rises less than 1g/, and the cell culture medium that wherein uses in the step c) comprises the recombinant albumin that rises greater than 1g/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant albumin that rises less than 2g/, and the cell culture medium that wherein uses in the step c) comprises the recombinant albumin that rises greater than 1g/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant albumin that rises less than 1g/, and the cell culture medium that wherein uses in the step c) comprises the recombinant albumin that rises greater than 2g/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant albumin that 0.5g/ at the most rises, and the cell culture medium that wherein uses in the step c) comprises the recombinant albumin that 2.5g/ at least rises.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant transferrin less than 5 μ g/ml, and the cell culture medium that wherein uses in the step c) comprises the recombinant transferrin greater than 5 μ g/ml.Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant transferrin less than 10 μ g/ml, and the cell culture medium that wherein uses in the step c) comprises the recombinant transferrin greater than 10 μ g/ml.Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant transferrin less than 5 μ g/ml, and the cell culture medium that wherein uses in the step c) comprises the recombinant transferrin greater than 10 μ g/ml.Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant transferrin less than 10 μ g/ml, and the cell culture medium that wherein uses in the step c) comprises the recombinant transferrin greater than 5 μ g/ml.Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises the recombinant transferrin of 4 μ g/ml at the most, and the cell culture medium that wherein uses in the step c) comprises the recombinant transferrin of minimum 6 μ g/ml.
Of the present invention preferred embodiment in; The cell culture medium that uses in the step a) comprises the recombinant albumin that rises less than 2g/ and less than the recombinant transferrin of 5 μ g/ml, and the cell culture medium that wherein uses in the step c) comprises the recombinant albumin that rises greater than 2g/ and greater than the recombinant transferrin of 5 μ g/ml.Of the present invention preferred embodiment in; The cell culture medium that uses in the step a) comprises the recombinant albumin that rises less than 1g/ and less than the recombinant transferrin of 10 μ g/l, and the cell culture medium that wherein uses in the step c) comprises the recombinant albumin that rises greater than 1g/ and greater than the recombinant transferrin of 10 μ g/ml.Of the present invention preferred embodiment in; The cell culture medium that uses in the step a) comprises the recombinant albumin that rises less than 1g/ and less than the recombinant transferrin of 5 μ g/ml, and the cell culture medium that wherein uses in the step c) comprises the recombinant albumin that rises greater than 2g/ and greater than the recombinant transferrin of 10 μ g/ml.Of the present invention preferred embodiment in; The cell culture medium that uses in the step a) comprises recombinant albumin that 0.5g/ at the most rises and the recombinant transferrin of maximum 4 μ g/ml, and the cell culture medium that uses in the step c) comprises recombinant albumin that 3g/ at least rises and the recombinant transferrin of minimum 15 μ g/ml.
Of the present invention preferred embodiment in, the recombinant albumin that the cell culture medium that the cell culture medium that uses in the step a) comprises than uses in the step c) lacks, especially BSA.Of the present invention preferred embodiment in, the recombinant transferrin that the cell culture medium that the cell culture medium that uses in the step a) comprises than uses in the step c) lacks, especially Transferrins,iron complexes.Of the present invention preferred embodiment in, recombinant albumin that the cell culture medium that the cell culture medium that uses in the step a) comprises than uses in the step c) lacks (especially BSA) and recombinant transferrin (especially Transferrins,iron complexes).
Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) is the substratum that does not contain animal component, more preferably not protein-contg substratum.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) is the substratum that does not contain animal component.
Of the present invention preferred embodiment in; Said method comprises that also step d) cultivates said cell, recombinant transferrin and/or recombinant albumin that the cell culture medium that said the 3rd serum-free cell culture medium comprises the concentration ratio step c) is low in the 3rd serum-free cell culture medium.Of the present invention preferred embodiment in; Said method comprises that also step d) cultivates said cell, recombinant transferrin and recombinant albumin that the cell culture medium that said the 3rd serum-free cell culture medium comprises the concentration ratio step c) is low in the 3rd serum-free cell culture medium.Of the present invention preferred embodiment in, said method comprises that also step d) cultivates said cell, the low recombinant transferrin of cell culture medium that said the 3rd serum-free cell culture medium comprises the concentration ratio step c) in the 3rd serum-free cell culture medium.Of the present invention preferred embodiment in, said method comprises that also step d) cultivates said cell, the low recombinant albumin of cell culture medium that said the 3rd serum-free cell culture medium comprises the concentration ratio step c) in the 3rd serum-free cell culture medium.
Of the present invention preferred embodiment in; Said method comprises that also step d) cultivates said cell in the 3rd serum-free cell culture medium; The recombinant transferrin that said the 3rd serum-free cell culture medium is comprised and the concentration of recombinant albumin be in the cell culture medium of step c) recombinant transferrin and recombinant albumin 80% or lower, preferred 50% or lower.
Of the present invention preferred embodiment in; Said method comprises that also step d) cultivates said cell in the 3rd serum-free cell culture medium, and recombinant transferrin and the cell culture medium of concentration ratio step c) that said the 3rd serum-free cell culture medium comprises low at least 2 times of the cell culture medium of concentration ratio step c) hang down at least 2 times recombinant albumin.
According to preferred embodiment, step d) is carried out after step c).
In preferred implementation of the present invention, the cell culture medium of step d) does not contain recombinant transferrin and recombinant albumin.
Of the present invention preferred embodiment in, said serum-free cell culture medium is the cell culture medium that does not contain animal component.Of the present invention preferred embodiment in, said serum-free cell culture medium is not protein-contg cell culture medium.
Of the present invention preferred embodiment in, the serum-free cell culture medium of step a) is the cell culture medium that does not contain animal component.Of the present invention preferred embodiment in, the serum-free cell culture medium of step a) is not protein-contg cell culture medium.
Of the present invention preferred embodiment in, the serum-free cell culture medium of step c) is the cell culture medium that does not contain animal component.Of the present invention preferred embodiment in, the serum-free cell culture medium of step c) is not protein-contg cell culture medium.
Of the present invention preferred embodiment in, the serum-free cell culture medium of step d) is the cell culture medium that does not contain animal component.Of the present invention preferred embodiment in, the serum-free cell culture medium of step d) is not protein-contg cell culture medium.
Of the present invention preferred embodiment in, the Osmolality of the cell culture medium of step c) (osomlality) is 280-320mOsmol/kg H 2O.
Of the present invention preferred embodiment in, the Osmolality of the cell culture medium of step c) is 280-300mOsmol/kg H 2O.Of the present invention preferred embodiment in, the pH of the cell culture medium of step c) is 6.0-8.0.Of the present invention preferred embodiment in, the pH of the cell culture medium of step c) is 6.8-7.1.
Of the present invention preferred embodiment in, the Osmolality of the cell culture medium of step c) is 280-320mOsmol/kg H 2O, pH are 6.8-7.1.
Of the present invention preferred embodiment in, the Osmolality of the cell culture medium of step c) is 280-300mOsmol/kg H 2O, pH are 6.8-7.0.Of the present invention preferred embodiment in, step a) and/or c) and/or the cell culture medium that d) uses comprise L-glutaminate.Of the present invention preferred embodiment in, step a) and/or c) and/or the cell culture medium that d) uses comprise the L-glutaminate of concentration less than 6mM.Of the present invention preferred embodiment in, step a) and/or c) and/or the cell culture medium that d) uses comprise the L-glutaminate of concentration less than 4mM.Of the present invention preferred embodiment in, step a) and/or c) and/or the cell culture medium that d) uses comprise the L-glutaminate of concentration less than 2mM.Of the present invention preferred embodiment in, step a) and/or c) and/or the cell culture medium that d) uses do not contain L-glutaminate.
Of the present invention preferred embodiment in, cell culture medium comprises L-glutaminate.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises L-glutaminate.Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) comprises L-glutaminate.Of the present invention preferred embodiment in, step a) and c) in the cell culture medium that uses comprise L-glutaminate.Of the present invention preferred embodiment in, step a), c) and d) in the cell culture medium that uses comprise L-glutaminate.
Of the present invention preferred embodiment in, the cell culture medium that step a) is used comprises the L-glutaminate of concentration less than 50mM.Of the present invention preferred embodiment in, the cell culture medium that step c) is used comprises the L-glutaminate of concentration less than 50mM.Of the present invention preferred embodiment in, the cell culture medium that step d) is used comprises the L-glutaminate of concentration less than 50mM.
Of the present invention preferred embodiment in, the cell culture medium that step a) is used comprises the L-glutaminate of concentration less than 6mM.Of the present invention preferred embodiment in, the cell culture medium that step c) is used comprises the L-glutaminate of concentration less than 6mM.Of the present invention preferred embodiment in, the cell culture medium that step d) is used comprises the L-glutaminate of concentration less than 6mM.
Of the present invention preferred embodiment in, the cell culture medium that step a) is used comprises the L-glutaminate of concentration less than 4mM.Of the present invention preferred embodiment in, the cell culture medium that step c) is used comprises the L-glutaminate of concentration less than 4mM.Of the present invention preferred embodiment in, the cell culture medium that step d) is used comprises the L-glutaminate of concentration less than 4mM.
Of the present invention preferred embodiment in, step a) and c) cell culture medium that uses comprises the L-glutaminate of concentration less than 50mM.Of the present invention preferred embodiment in, step a) and c) cell culture medium that uses comprises the L-glutaminate of concentration less than 6mM.Of the present invention preferred embodiment in, step a) and c) cell culture medium that uses comprises the L-glutaminate of concentration less than 4mM.Of the present invention preferred embodiment in, step a), c) and the cell culture medium that d) uses comprise the L-glutaminate of concentration less than 4mM.
Of the present invention preferred embodiment in, employed all cells substratum all comprises the L-glutaminate of same concentrations.Of the present invention preferred embodiment in, employed all cells substratum all comprises the L-glutaminate of concentration less than 50mM.Of the present invention preferred embodiment in, employed all cells substratum all comprises the L-glutaminate of concentration less than 20mM.Of the present invention preferred embodiment in, employed all cells substratum all comprises the L-glutaminate of concentration less than 6mM.Of the present invention preferred embodiment in, employed all cells substratum all comprises the L-glutaminate of concentration less than 4mM.Of the present invention preferred embodiment in, step a) and/or c) in the cell culture medium that uses comprise iron.Of the present invention preferred embodiment in, step a) and/or c) and/or d) in the cell culture medium that uses comprise iron.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises iron.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises iron.Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) comprises iron.Of the present invention preferred embodiment in, step a) and c) in the cell culture medium that uses comprise iron.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) comprises non-Tf-Fe.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises non-Tf-Fe.Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) comprises non-Tf-Fe.
Of the present invention preferred embodiment in, step a) and/or c) in the cell culture medium that uses comprise non-Tf-Fe.Of the present invention preferred embodiment in, step a) and/or c) and/or d) in the cell culture medium that uses comprise non-Tf-Fe.
Of the present invention preferred embodiment in, step a) and c) in the cell culture medium that uses comprise non-Tf-Fe.Of the present invention preferred embodiment in, step a), c) and d) in the cell culture medium that uses comprise non-Tf-Fe.
Of the present invention preferred embodiment in, culturing cell at least 1 day in step a) and the said cell of not subculture.Of the present invention preferred embodiment in, culturing cell at least 2 days in step a) and the said cell of not subculture.Of the present invention preferred embodiment in, culturing cell at least 3 days in step a) and the said cell of not subculture.
Of the present invention preferred embodiment in, culturing cell is at least 5 days in step c).Of the present invention preferred embodiment in, culturing cell is at least 10 days in step c).Of the present invention preferred embodiment in, culturing cell at least 5 days in step c) and the said cell of not subculture.Of the present invention preferred embodiment in, culturing cell at least 10 days in step c) and the said cell of not subculture.
Of the present invention preferred embodiment in, culturing cell is at least 3 days in step d).Of the present invention preferred embodiment in, culturing cell is at least 6 days in step d).
Of the present invention preferred embodiment in, culturing cell at least 3 days in step d) and the said cell of not subculture.Of the present invention preferred embodiment in, culturing cell at least 6 days in step d) and the said cell of not subculture.
Of the present invention preferred embodiment in, at step c) and d) in total co-cultured cell at least 3 days.Of the present invention preferred embodiment in, at step c) and d) in total co-cultured cell at least 6 days.
Of the present invention preferred embodiment in, at step c) and d) in total co-cultured cell at least 3 days and the said cell of not subculture.Of the present invention preferred embodiment in, at step c) and d) in total co-cultured cell at least 6 days and the said cell of not subculture.
Of the present invention preferred embodiment in, the volume of culture in the step a) is 0.1ml, more preferably 0.5ml, more preferably 3ml, more preferably 15ml, more preferably 100ml.
Of the present invention preferred embodiment in, the volume of culture in the step a) is 0.1ml at least.Of the present invention preferred embodiment in, the volume of culture in the step a) is 0.5ml at least.Of the present invention preferred embodiment in, the volume of culture in the step a) is 3ml at least.Of the present invention preferred embodiment in, the volume of culture in the step a) is about 3ml.Of the present invention preferred embodiment in, the volume of culture in the step a) is 15ml at least.Of the present invention preferred embodiment in, the volume of culture in the step a) is about 15ml.Of the present invention preferred embodiment in, the volume of culture in the step a) is 100ml at least.Of the present invention preferred embodiment in, the volume of culture in the step a) is about 100ml.Of the present invention preferred embodiment in, the volume of culture in the step a) is 1 liter at the most.Of the present invention preferred embodiment in, the volume of culture in the step a) is 0.5 liter at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is about 5ml at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is 2ml at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is about 2ml at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is about 1ml at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is 450 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is 150 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is 100 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is about 100 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is 30 μ l.Of the present invention preferred embodiment in, the volume of culture in the step c) is 10 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step c) is about 10 μ l at the most.
Of the present invention preferred embodiment in, the volume of culture in the step c) is at least 1 μ l.Of the present invention preferred embodiment in, the volume of culture in the step c) is at least 5 μ l.Of the present invention preferred embodiment in, the volume of culture in the step c) is at least about 5 μ l.Of the present invention preferred embodiment in, the volume of culture in the step c) is at least about 10 μ l.Of the present invention preferred embodiment in, the volume of culture in the step c) is at least 100 μ l.Of the present invention preferred embodiment in, the volume of culture in the step c) is at least about 100 μ l.Of the present invention preferred embodiment in, the volume of culture in the step c) is at least about 0.5ml.Of the present invention preferred embodiment in, the volume of culture in the step c) is 0.5ml at least.Of the present invention preferred embodiment in, the volume of culture in the step c) is at least about 1ml.Of the present invention preferred embodiment in, the volume of culture in the step d) is 20 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step d) is 60 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step d) is 200 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step d) is 600 μ l at the most.Of the present invention preferred embodiment in, the volume of culture in the step d) is 1.8ml.Of the present invention preferred embodiment in, the volume of culture in the step d) is 5ml at the most.
Of the present invention preferred embodiment in, the volume of culture in the step d) is at least 1 μ l.Of the present invention preferred embodiment in, the volume of culture in the step d) is at least about 1 μ l.Of the present invention preferred embodiment in, the volume of culture in the step d) is at least 10 μ l.Of the present invention preferred embodiment in, the volume of culture in the step d) is at least about 10 μ l.Of the present invention preferred embodiment in, the volume of culture in the step d) is at least 50 μ l.Of the present invention preferred embodiment in, the volume of culture in the step d) is at least 100 μ l.Of the present invention preferred embodiment in, the volume of culture in the step d) is at least 300 μ l.Of the present invention preferred embodiment in, the volume of culture in the step d) is at least about 1.5ml.Of the present invention preferred embodiment in, the volume of culture in the step d) is at least about 3ml.Of the present invention preferred embodiment in, the volume of culture in the step d) is 3ml at least.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 50mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 200mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 1000mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 2000mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 5000mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 50000mg/ at the most rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 0.1mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 1mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 5mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 50mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 250mg/ at least rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant transferrin that 2500mg/ at the most rises.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 50mg/ at least rises and the recombinant transferrin of 0.1mg/ liter at least.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 1000mg/ at least rises and the recombinant transferrin of 1mg/ liter at least.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 2000mg/ at least rises and the recombinant transferrin of 5mg/ liter at least.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) comprises the recombinant albumin that 5000mg/ at least rises and the recombinant transferrin of 50mg/ liter at least.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) comprises the recombinant albumin that rises less than 5000mg/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) comprises the recombinant transferrin that rises less than 50mg/.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) comprises recombinant albumin that rises less than 5000mg/ and the recombinant transferrin that rises less than 50mg/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) comprises recombinant albumin that rises less than 2000mg/ and the recombinant transferrin that rises less than 5mg/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) comprises recombinant albumin that rises less than 50mg/ and the recombinant transferrin that rises less than 0.1mg/.Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) does not contain recombinant albumin and does not contain recombinant transferrin.
Of the present invention preferred embodiment in, the cell culture medium that uses in the step a) is the substratum that does not contain animal component.Of the present invention preferred embodiment in, the cell culture medium that uses in the step c) is the substratum that does not contain animal component.Of the present invention preferred embodiment in, the cell culture medium that uses in the step d) is the substratum that does not contain animal component.
Of the present invention preferred embodiment in, step a) and c) in the cell culture medium that uses be the substratum that does not contain animal component.Of the present invention preferred embodiment in, step c) and d) in the cell culture medium that uses be the substratum that does not contain animal component.Of the present invention preferred embodiment in, step a), c) and d) in the cell culture medium that uses be the substratum that does not contain animal component.
Of the present invention preferred embodiment in, all cells substratum of use all is the substratum that does not contain animal component.Of the present invention preferred embodiment in, in step b), reduce cell mass through the automated cell separation system.
Of the present invention preferred embodiment in, in step b), separate unicellular through the automated cell separation system.Of the present invention preferred embodiment in, in step b), separate unicellular through FACS.
Of the present invention preferred embodiment in, the cell of cell mass does not contain feeder cell.
Of the present invention preferred embodiment in, the cell of cell mass is an eukaryotic cell.Of the present invention preferred embodiment in, the cell of cell mass is a mammalian cell.Of the present invention preferred embodiment in, the cell of cell mass is people's cell.Of the present invention preferred embodiment in, the cell of cell mass is a zooblast.Of the present invention preferred embodiment in, the cell of cell mass is not a human embryo stem cell.
Of the present invention preferred embodiment in, cell mass is a clone.Of the present invention preferred embodiment in, cell mass is Chinese hamster ovary celI system, NS0 clone, Per.C6 clone, HEK293 clone or bhk cell system.Of the present invention preferred embodiment in, cell mass is a Chinese hamster ovary celI system.Of the present invention preferred embodiment in, cell mass is a clone.Of the present invention preferred embodiment in, cell mass is a NS0 clone.Of the present invention preferred embodiment in, cell mass is a clone.Of the present invention preferred embodiment in, cell mass is a Per.C6 clone.Of the present invention preferred embodiment in, cell mass is a clone.Of the present invention preferred embodiment in, cell mass is a HEK293 clone.Of the present invention preferred embodiment in, cell mass is a clone.Of the present invention preferred embodiment in, cell mass is a bhk cell system.Of the present invention preferred embodiment in, the cell of cell mass is a prokaryotic cell prokaryocyte.
In preferred embodiment, the invention still further relates to the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin and be used for the purposes of culturing cell concentration less than the cell mass of about 100 cell/ml.In preferred embodiment, the invention still further relates to the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin and be used for the purposes of culturing cell concentration less than the cell mass of 100 cell/ml.
In preferred embodiment, the invention still further relates to the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin and be used to cultivate single celled purposes.
The preferred implementation that relates to the inventive method also is the embodiment that is preferred for purposes of the present invention.
In preferred embodiment, the invention still further relates in the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin cultured cells concentration less than the cell mass of about 100 cell/ml.
In preferred embodiment, the invention still further relates in the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin cultured cells concentration less than the cell mass of 100 cell/ml.
In preferred embodiment, the invention still further relates in the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin, cultivate unicellular.What relate to the inventive method preferred embodiment also is to be used for the preferred implementation that contains the serum-free cell culture medium cultured cells crowd of recombinant albumin and recombinant transferrin of the present invention.
The present invention is the serum free medium that is used for single cell clone that contains recombinant albumin and recombinant transferrin.Utilization has adapted to the Chinese hamster ovary celI system of in the substratum that does not contain serum, growth factor, albumen and peptone, growing and has made an experiment.Said substratum comprises inorganic source of iron.In this substratum, clone cell concn in batch feeding process reaches 3 * 10 7Individual cell/ml.This has proved the extraordinary growth that does not rely on serum and albumen additive.Yet the clonal growth rate of same cell in same medium that is in unicellular attitude is very low.
All of a sudden, the contriver finds through adding the clonal growth that recombinant protein can significantly strengthen clone.Fact proved that with the growth phase ratio in the cell mass, clone has different requirement to substratum when growing with unicellular attitude.Those skilled in the art considered that the clone that does not rely on the growth of serum and albumen also should be in serum-free and proteic environment clonal growth.The contriver is surprised to find that when cultivating with unicellular attitude and in cell mass, cultivating, same clone has different requirement to substratum.
Therefore, all of a sudden only must in step c), have recombinant albumin and/or recombinant transferrin.Said cell before step c) with after do not need (not needing high density at least) recombinant albumin and/or recombinant transferrin.
According to the present invention, when the needs clone cell is grown, with clone is contacted with the substratum that contains recombinant albumin and recombinant transferrin.Obviously, recombinant albumin and recombinant transferrin pair cell have synergy.Also separated measuring two kinds of albumen.They are all with low-level promotion cell proliferation.When two kinds of albumen of combination, almost perhaps even than the level that contains serum contrast culture object height significantly improve single celled cell growth and survival with the level that contains the serum control cultures.
Whether other parameter that the contriver further detects substratum also plays a role in improving clonal growth.The contriver is surprised to find that unicellular also have different requirement aspect culture medium Osmolality and the medium pH.When reducing the Osmolality of substratum, even can improve clonal growth more.The preferred weight osmolarity of substratum is 260-360mOsmol/kg H 2O, more preferably 270-340mOsmol/kg H 2O, more preferably 280-320mOsmol/kg H 2O, more preferably 290-300mOsmol/kg H 2O.When the pH of substratum is lower than common substratum, even can improve clonal growth more.The pH of preferred substratum is 6.7-7.3, more preferably 6.8-7.2, more preferably 6.8-7.0.
Recombinant protein is expensive as medium additives.Therefore, it is significant in substratum, omitting recombinant protein.According to preferred implementation of the present invention, promote the simple of clonal growth and have more cost-benefit mode be in the first step in the substratum that contains lower concentration recombinant albumin and recombinant transferrin perhaps culturing cell in the substratum that does not contain recombinant albumin and recombinant transferrin of culturing cell.In second step, make the cell recombinant albumin high and the substratum exposing cell of recombinant transferrin with containing the concentration that adopts in the concentration ratio the first step.In the 3rd step, can in the substratum of recombinant albumin with low concentration and recombinant transferrin, perhaps can omit recombinant albumin and recombinant transferrin fully by culturing cell.In fact through this process; Can be only adopt recombinant albumin and recombinant transferrin, or can omit expensive recombinant protein in the single cell clone step with in all other cell culture step and can set up and have cost benefit and simple method is used for single cell clone through the cell concn that reduces.
Another major advantage of method of the present invention is, can culturing cell in the substratum that does not contain animal component in steps, this supervision department for manufacture of therapeutic protein has significant advantage.
Term " recombinant protein " refers to the albumen by the nucleic acid encoding that is introduced into host cell.The said nucleic acid of said host cell expression." albumen " that this paper uses broadly refers to polyamino acid, for example peptide, polypeptide, albumen, lipoprotein, gp etc.
Term " serum free medium " or " serum-free cell culture medium " are the substratum that does not contain the serum (for example foetal calf serum (FBS), Ox blood serum, horse serum, lowlenthal serum, human serum etc.) from any biology.
Term " cell cultures " or " cultivation " expression cell remain in the external environment of artificial; Yet should understand; Term " cell cultures " is upperseat concept and can be used for not only containing individual cells or the only unicellular or only cultivation of cell mass; But also the cultivation of cover tissue or organ, to this, term " tissue culture " or " organ culture " sometimes can be exchanged with term " cell cultures " and used.
Term " contact " refers to that culturing cell places contains the culturing bottle that said cell waits to be incubated at substratum wherein with external treating.Term " contact " is contained cell is mixed with substratum, substratum is moved on the cell in liquid to the culturing bottle, and cell is immersed in the substratum.
Term " BSA " refers to that as the albumen that is included in the Abundant protein in the blood plasma, it promotes the maintenance of Osmolality in the blood and possibly combine with nutrient so that these materials are sent to cell.There is multi-form BSA, divides or the level of part, BSA is divided or part such as but not limited to the level of human serum albumin (HSA), bovine serum albumin (BSA), HSA.Regulate relevant with the Osmolality of substratum and combines with nutrient or nutrient to be transferred to cell relevant, BSA can be other any albumen or the polypeptide that aspect cell growth, cell survival or cells produce power, produces basically similar result.Preferably, said BSA is that the people originates.Most preferably, said BSA is a human serum albumin.Even more preferably, said BSA is the human serum albumin (" reorganization HSA ") that reorganization produces.Said reorganization HSA can produce in multiple biology, for example prokaryotic cell prokaryocyte or eukaryotic cell, for example bacterium, plant or yeast etc.Known in the art multiple host, for example produce recombinant albumin among intestinal bacteria (EP73646) and the fungal cell (WO0044772).Such as but not limited to, said recombinant albumin is people's recombinant albumin that Sigma (A7223) describes.
Term " Transferrins,iron complexes " refers to have any biological compound of iron combination or sequestering power.The instance of Transferrins,iron complexes includes but not limited to have iron avidity, and for example iron combines or any albumen, polypeptide or the peptide of sequestering power.Other instance of Transferrins,iron complexes includes but not limited to have with the cell TfR albumen, polypeptide or the peptide of any avidity.This albumen, polypeptide or peptide can be discerned, part combines or combine fully the cell TfR.Can make Transferrins,iron complexes saturated or make Transferrins,iron complexes saturated with iron without iron.If make Transferrins,iron complexes saturated without iron, then substratum can contain inorganic iron.If make Transferrins,iron complexes saturated with iron, then said substratum can not contain inorganic iron or said substratum can comprise other inorganic iron.Preferably, make Transferrins,iron complexes saturated with iron.More preferably, Transferrins,iron complexes is the saturated HTrf of iron.
Term " recombinant transferrin " is the Transferrins,iron complexes that reorganization produces in any biology.Said recombinant transferrin can for example produce in prokaryotic cell prokaryocyte or the eukaryotic cell at multiple biology.Recombinant transferrin can produce in bacterium, plant, fungi or yeast.The HTrf who preferably recombinates and produce.Such as but not limited to, the recombinant human Transferrins,iron complexes is the recombinant human Transferrins,iron complexes of in product description, describing from Millipore (9701-10).
In especially preferred embodiment, the present invention relates to substratum, the concentration of the recombinant albumin that comprises in the wherein said substratum is for be preferably greater than the 0.1g/ liter at least; At least be preferably greater than the 0.2g/ liter, be preferably greater than the 0.5g/ liter at least, be preferably greater than the 1.0g/ liter at least; At least be preferably greater than the 2g/ liter, perhaps in especially preferred embodiment, be preferably greater than the 3g/ liter at least; Perhaps in further especially preferred embodiment, be preferably greater than the 5g/ liter at least.In further preferred embodiment, the invention provides above-mentioned substratum, the concentration of wherein said recombinant transferrin is for be preferably greater than the 0.1mg/ liter at least; At least be preferably greater than the 1mg/ liter, be preferably greater than the 2mg/ liter at least, be preferably greater than the 4mg/ liter at least; At least be preferably greater than the 6mg/ liter, be preferably greater than the 8mg/ liter at least, be preferably greater than the 10mg/ liter at least; At least be preferably greater than the 20mg/ liter, be preferably greater than the 50mg/ liter at least, be preferably greater than the 200mg/ liter at least; Perhaps in especially preferred embodiment, be preferably greater than the 1000mg/ liter at least.
Term " does not contain animal component " and refers to wherein not use the substratum or the cell culture processes of the composition that originates from the animal or human.
Term " is enough to support unicellular growth ", and the said substratum of expression can be supported single celled growth, but does not require that said substratum is actually used in the unicellular growth of support.Said substratum can be suitable for single celled growth or be used for concentration less than the growth of the cell mass of 100 cell/ml or be used for the growth of cell concn greater than the cell mass of 100 cell/ml.Can continue clonal growth,, for example under the growth conditions less than the growth of the density of about 100 cell/ml, substratum of the present invention contacted with comprising single celled cell mass promptly with low-down cell density.
Term " cell culture medium ", " tissue culture medium (TCM) ", " substratum ", " deposit substratum " " clone's substratum " refer to be used for the nutrient solns of culturing cell or tissue, and can exchange use.Substratum is the medium that is fit to cultivate or be fit to a cell of incubation or several cells.This substratum can comprise the nutrient that is used to keep cell integrity or cell viability or cell growth or cells produce power.The preferred liquid substratum.Especially preferred substratum is described among the WO 2007/036291 and can be used for the present invention.Especially preferred substratum comprises all that be used for cell growth, cell viability and cells produce power must material.Especially preferred substratum can comprise, such as but not limited to glucose, amino acid, salt, trace element and lipid acid.
The cell cultures of mammalian cell is the routine operation of describing in detail in science textbook and the handbook at present.Be shown in R.lan Fresney for example, Culture of Animal cells, a manual, 4thedition, Wiley-Liss/N.Y., 2000 in detail.The substratum and the cultural method (for example being used for mammal cell line) that use with cultivation additive combination of the present invention itself are well known in the art.This substratum preferably is made up of solvent (for example water), carbon source, nitrogenous source, amino acid, pH regulator agent, trace element, lipid acid, Nucleotide.The cell culture medium that to be used for preferred substratum of the present invention be standard; It also can adapt to the needs of particular cell types; And include but not limited to Roswell Park Memorial Institute (RPMI) 1640 substratum, L-15 substratum, DMEM (Dulbecco ' smodified Eagle ' s medium, DMEM), the minimum dulbecco minimum essential medium Dulbecco of Iger (MEM), breathe out nurse F12 substratum (Ham ' s F12medium) or Yi Si koff improvement DMEM.Other preferred culture medium is for example to be designed for Kazakhstan nurse F10 or F12 substratum that Chinese hamster ovary celI is cultivated especially.Be used for other preferred substratum of the present invention and be adapted to the Chinese hamster ovary celI cultivation especially, and be described in for example EP 0 481 791.Being used for preferred substratum of the present invention can also be commercially available substratum, such as but not limited to CD CHO (Gibco, 10743), ProCHO5 (BioWhittaker, BE 12-766Q), HyQSFM4CHO (HyClone, SH30548.02).
In further preferred embodiment of the present invention, said substratum can comprise L-glutaminate, and Stimulina can be used the Stimulina surrogate wholly or in part, and for example GLUTAMAX (GIBCO Cat.Nr:35050-061) replaces.In further preferred embodiment of the present invention, said substratum can comprise the L-glutaminate of lower concentration.The concentration of L-glutaminate can be the 900mg/ liter to the maximum, preferred 600mg/ liter, more preferably 300mg/ liter, more preferably 100mg/ liter, more preferably 50mg/ liter, more preferably 20mg/ liter.Said substratum can not contain L-glutaminate.When substratum does not contain L-glutaminate, be particularly useful for selecting the cell of gene transformation with glutamine synthetase.
In further preferred embodiment of the present invention, said substratum comprises at least a glucide source.Preferred working concentration is the glucose that 0-10g/ rises.Can use other glucide, replace glucose wholly or in part such as but not limited to fructose, seminose, semi-lactosi.
In further preferred embodiment of the present invention, said substratum can not contain any inorganic source of iron and/or not contain any iron chelating compounds.
Alternatively, in one embodiment, preferred substratum of the present invention can also comprise from animal-origin, from plant origin or from the zymic glucide.Preferably from the hydrolysate of plant origin, for example soy peptone or yeast.
In particularly preferred embodiments, substratum of the present invention does not contain serum.In particularly preferred embodiments, substratum of the present invention does not contain the product of direct or indirect separation from the animal source.Of the present invention especially preferred embodiment in, said substratum does not contain animal component.Of the present invention especially preferred embodiment in, said substratum does not contain hydrolysate.
In further preferred embodiment of the present invention, said substratum can comprise inorganic iron.
In further preferred embodiment of the present invention, said substratum can comprise one or more iron chelating compounds.
Term " iron " is intended to represent that nucleidic mass is 55,845 transition-metal Fe.Term iron is upperseat concept, and it comprises and contains one or more iron ions (Fe for example 3+And/or Fe 2+Ion) all molecules.Fe 3+And/or Fe 2+Ion can exist with the molysite form.Molysite can hydration and or dehydration.In particularly preferred embodiments, said source of iron comprises Fe (II) and/or Fe (III) ion.In particularly preferred embodiments, the source of iron that is used for the present invention is selected from the group of being made up of following: tertiary iron phosphate (III), ferric pyrophosphate (III), iron nitrate (III), ferric sulfate (II), iron(ic)chloride (III), ironic lactate (II), ironic citrate (III), ammonium citrate iron (III), Iron Dextran and iron edta sodium salt or its hydration or dehydrated form.
Iron can also with another kind of molecule (for example carrier or sequestrant) complexing.Some preferred especially limiting examples with the complexing iron of sequestrant are iron lactate (II) hydrate, ironic citrate (III), ammonium citrate iron (III), Iron Dextran and iron edta sodium salt.Said iron can also be alternatively and following other molecular complex: for example US 6; 048; The molecule of describing in 728, i.e. pyridoxal base Vanizide, Citrate trianion, acetylacetonate and multiple other organic acid are like oxysuccinic acid, succsinic acid, fumaric acid and α-Tong Wuersuan.Be used for other iron chelating agent of the present invention and be shown in WO 2001/016294.
The invention is not restricted to the cell of any kind.The instance of cell type comprises mammalian cell, insect cell, bacterial cell and yeast cell.Said cell can also be primary cell or stem cell.Said cell is not transformed or the naturally occurring cell of transfection.Said cell can also be with the one or more carrier transfection of recombinant gene expression or reconstitution cells of conversion of being used for.Can transform said cell with the virus that is used to produce spawn (for example viral product).Said cell can originate from hamster, mouse, people or any other animal.Said cell can also be a clone, such as but not limited to Chinese hamster ovary celI, CHO K1 cell, CHO DUKX cell, CHO DG44 cell, NS0 cell, Per.C6 cell, bhk cell, SP2/0 cell, HEK293 cell.
Further preferred embodiment of the present invention is the theme of dependent claims.
Following examples and accompanying drawing are described the present invention in more detail.
Fig. 1 is presented at the comparison of cloning efficiency among conditioned medium and the rHSA.
Fig. 2 shows the comparison that utilizes the cloning efficiency that is with or without the RHA that adds the recombinant human Transferrins,iron complexes.
Fig. 3 has shown the comparison of the cloning efficiency that utilizes rHSA and rHTR and their combination separately.
Fig. 4 has shown the titration of the recombinant human Transferrins,iron complexes that adds and do not add RHA and the cloning efficiency that is produced.
Fig. 5 shows that the combination of rHSA and rHTR is added on and promotes the effect of the sedimentary single celled cell of FACS institute in growing.In addition, these results have shown the validity that this culture medium prescription to different Chinese hamster ovary celIs is.
Embodiment
Cell
All use following three kinds of Chinese hamster ovary celIs to be for all experiments:
1) DHFR (Tetrahydrofolate dehydrogenase) defective type CHO DG44 host cell system (Urlaub and Chasin, Proc.Natl.Acad.Sci., 1980,77:4216).
2) by 1) in the clone mentioned produce the conversion CHODG44 subclone (called after clone23) of expressing DHFR and recombinant monoclonal antibodies simultaneously.This clone transforms CHO DG44 host cell system through the carrier that DHFR and IgG antibody gene are arranged with load to produce.Utilize the MTX transfectant that in substratum, increases.
3) CHO K1 host cell system (Puck TT etc., J.Exp.Med., 1958,108:945-956).The all cells that the present invention adopts does not all require to have in the substratum and is used to any albumen of growing and surviving.All cells system does not all rely on serum, albumen, growth factor, hydrolysate, BSA and Transferrins,iron complexes and grows.
Cell culture condition
The deposit culturing cell that will be used for single cell clone remains on and shakes bottle or revolving bottle.With 3 * 10 5In shaking bottle, and every batch of cell concn reaches 5 * 10 to the cell concn of individual cell/ml after 2-4 days vegetative period with cell inoculation 5Individual cell/ml to 100 * 10 5Individual cell/ml.Stock culture was divided to fresh culture in every 2-4 days.This expression utilizes the small amounts of cells culture as inoculum and be transferred in the new bottle and be supplemented with fresh culture.When cell does not reach high density, centrifugal to it in the process of minute cell.With cell in incubator in 37 ℃, 7.5%CO 2Cultivate under the atmosphere.Cultivate number generation by this way.A generation is defined as 2-4 days during cultivation.Take out the cell that is used for the single cell clone experiment from these stock culture that are in exponential phase of growth.
Substratum
The deposit substratum
In said experiment, use two kinds of different deposit substratum.First; Commercially available deposit substratum (CD CHO with unknown prescription; Gibco, 10743), second; Privately owned deposit substratum with following characteristic: said privately owned deposit substratum contains all that be useful on cell growth, cell survival and cells produce power must material, such as but not limited to glucose, amino acid, salt, trace element and lipid acid.Said privately owned deposit substratum does not contain serum, albumen, growth factor and peptone.Said privately owned deposit substratum does not contain animal component.Said privately owned deposit substratum comprises the inorganic source of iron that is used to the additional iron of cell.Said privately owned deposit substratum does not contain recombinant albumin and does not contain recombinant transferrin.Said privately owned deposit substratum is supplemented with the 6mM L-glutaminate before use.The Osmolality of said privately owned substratum is 330mOsmol/kg H 2O, pH are 7.2.
Promote the clone cell growth in clone's substratum of the deposit substratum that adopts not relying on, this proves that said deposit culture medium prescription is inoperative for the clone cell growth.
Clone's substratum
Adopt two kinds of different clone's substratum.First; Commercially available substratum (CDCHO with unknown prescription; Gibco, 10743), second; Privately owned clone's substratum with following characteristic: said privately owned clone's substratum contains all that be useful on cell growth, cell survival and cells produce power must material, such as but not limited to glucose, amino acid, salt, trace element and lipid acid.Said privately owned clone's substratum does not contain serum, albumen, growth factor and and peptone.Said privately owned clone's substratum does not contain animal component.Said privately owned clone's substratum comprise concentration be the inorganic source of iron tertiary iron phosphate (III) that rises of 2mg/ (Sigma, F1523).Said privately owned clone's substratum does not contain recombinant albumin and does not contain recombinant transferrin.Said privately owned clone's substratum is supplemented with the 2mM L-glutaminate before use.The Osmolality of said privately owned clone's substratum is 290mOsmol/kg H 2O, pH are 6.9.
In two kinds of clone's substratum, promote the clone cell growth, this proves that said deposit culture medium prescription is inoperative to the clone cell growth.Yet, as mentioned below, to the clone cell importantly inventive step of growing, for example use recombinant protein to replenish clone's substratum.
Carry out single cell clone through restricted dilution
Through from greater than 3 * 10 5The cell suspending liquid of the cell density dilution stock culture of the cell density to 4 of individual cell/ml cell/ml (=0.6 cell/150 μ l) carries out artificial restricted dilution (LD).With 1: 10 gradient with cell dilution in clone's substratum, be 1: 20 in each final dilution gradient of replenishing in clone's substratum.Be pumped in the 96-hole U base plate
Figure BPA00001444923100261
that every hole contains 150 μ l substratum with 0.6 cell in every hole.0.6 cells/well of numeral is the inoculating cell density on the statistics.In fact, when after inoculation during with microscope monitoring culture plate, some holes do not contain cell and some holes contain 2 or many cells more.
Carry out single cell clone through FACS
Through with carrying out single cell clone (SCC) in 1 the direct sorting of cell to 96-hole, every hole U base plate
Figure BPA00001444923100262
through FACS (cell sorting of fluorescent activation).Every hole 150 μ l clone substratum is provided to flat board.Before cell sorting, will have flat board incubation in incubator of substratum.Utilization is equipped with automatic cytological sedimentation unit (ACDU)
Figure BPA00001444923100263
(BD Biosciences) and carries out the FACS sorting with unicellular minute lectotype.
Incubation and evaluation
After carrying out SCC, immediately flat board is transferred in the incubator (37 ℃, 7.5%CO 2).Estimated successfully clone's number of amplification through naked eyes with through microscope in back 14 days in unicellular inoculation.The result representes with the % cloning efficiency.100% cloning efficiency of each experiment is set at clone's number of in the parallel positive control that carries out that is supplemented with 10%FBS (
Figure BPA00001444923100264
hot deactivation), growing.Because cell always is not in the isometric growth stage when being used for SCC, between different experiments, has the difference of cloning efficiency.Yet the inner difference of experiment is not twisted the whole observations that obtains through said research.
Embodiment 1: the comparison of the rHSA of the medium additives of conditioned medium and the unicellular growth of conduct promotion
The purpose of experiment is to estimate when with extremely low cell concn (for example unicellular attitude) inoculating cell culture, conditioned medium or whether can promote cell to grow as the RHA of the additive in the substratum.
CHO Clone23 cell is through material and artificial clone unicellular of the described restricted dilution of method part.For every kind of substratum combination, all spread two 96 orifice plates.Experimentize according to following:
Positive control (FBS): said clone's culture medium supplemented have 10% from
Figure BPA00001444923100271
the qualified foetal calf serum of hot deactivation (FBS) (10100-147).This expression 40ml clone culture medium supplemented has 4ml FBS (100% stock solution).
Conditioned medium K1: by CHO-K1 culture preparation condition substratum.The CHO-K1 cell is cultivated in shaking bottle in batch.Through two centrifugation step from CHO-K1 cellular segregation conditioned medium.At first, at room temperature with 190 * g eccentric visual cell suspension-s 3 minutes.Then supernatant is transferred in the new bottle, and at room temperature with 3000 * g recentrifuge 10 minutes.Subsequently, make through 0.2 μ m filter (
Figure BPA00001444923100272
PALL) filtration substratum.Utilize 50% prepared conditioned medium to dilute fresh clone's substratum.This expression makes the fresh clone's substratum of 20ml mix with the 20ml conditioned medium.
The interpolation of RHA (rHSA): utilizing RHA (rHSA) stock solution to replenish said clone's substratum to final concentration is the 2g/ liter.This expression, the fresh clone's culture medium supplemented of 40ml has the 50g/ of 1.6ml rHSA to rise stock solution (
Figure BPA00001444923100273
A7223).
According to the clone that the description incubation is dull and stereotyped and counting is produced in material and method part.
The result proves that (Fig. 1) conditioned medium is not enough to support clonal growth.RHSA supports clonal growth, but cloning efficiency is not enough when comparing with control cells.
Embodiment 2: add or do not add the test of the RHA of recombinant human Transferrins,iron complexes
The purpose of experiment is to estimate whether possibly improve cloning efficiency through adding the recombinant human Transferrins,iron complexes.
CHO Clone23 cell is through material and artificial clone unicellular of the described restricted dilution of method part.For every kind of substratum combination, all spread two 96 orifice plates.Experimentize according to following:
Positive control (FBS): said clone's culture medium supplemented have 10% from
Figure BPA00001444923100274
heat-inactivated qualified foetal calf serum (FBS) (10100-147).This expression 40ml clone culture medium supplemented has 4ml FBS (100% stock solution).
Negative control (not having to replenish): utilize to have no the pure clone's substratum of 40ml that further replenishes.
The interpolation of RHA (rHSA): utilizing RHA (rHSA) to replenish said fresh clone's substratum to final concentration is the 2g/ liter.This expression, the fresh clone's culture medium supplemented of 40ml has the 50g/ of 1.6ml rHSA to rise stock solution ( A7223).
The interpolation of RHA and recombinant human Transferrins,iron complexes (rHSA+rHTR): utilizing RHA (rHSA) to replenish said fresh clone's substratum to final concentration is the 2g/ liter, and further to be supplemented to final concentration with recombinant human Transferrins,iron complexes (rHTR) be the 5mg/ liter.This expression, the 20g/ that clone's culture medium supplemented that 40ml is fresh has the 50g/ of 1.6ml rHSA to rise stock solution and 10 μ l rHTR rises stock solution (
Figure BPA00001444923100282
(9701-10)).
According to the clone that the description incubation is dull and stereotyped and counting is produced in material and method part.
The result proves (Fig. 2), has no additional (negative control), does not see the clone.When fresh clone's culture medium supplemented to final concentration is 2g/ when rising rHSA, the clone of growth is about 20% of contrast.All of a sudden, when replenishing said clone's substratum with rHSA and rHTR, the clone's of growth quantity is increased to 80% of positive control.What is interesting is that in the deposit culturing process, the CHOClone23 clone of using in this experiment is cultivated in having no proteic substratum.Said deposit substratum comprises inorganic source of iron, and the good growth in deposit is cultivated is that cell does not rely on proteic evidence.Data show, even said cell does not need rHSA and rHTR in cell mass, but it needs these recombinant proteins with better growth in unicellular attitude.These results show that high clonal growth can be only because the interpolation of rHTR.Therefore, what is interesting is that observing rHSA does not influence unique interpolation of rHTR (referring to embodiment 3).
Embodiment 3: estimate the influence of rHSA and rHTR respectively
The purpose of experiment be respectively with combined test RHA (rHSA) and recombinant human Transferrins,iron complexes (rHTR) to observe each proteic effect respectively.Further purpose is to estimate two kinds of albumen whether to have synergy really.
CHO Clone23 cell is through material and artificial clone unicellular of the described restricted dilution of method part.For every kind of substratum combination, all spread two 96 orifice plates.Experimentize according to following:
Positive control (FBS): said clone's culture medium supplemented have 10% from
Figure BPA00001444923100283
heat-inactivated qualified foetal calf serum (FBS) (10100-147).This expression 40ml clone culture medium supplemented has 4ml FBS (100% stock solution).
The interpolation of RHA (rHSA): utilize rHSA to replenish said fresh clone's substratum to final concentration and be the 2g/ liter.This expression, the fresh clone's culture medium supplemented of 40ml has the 50g/ of 1.6ml rHSA to rise stock solution (
Figure BPA00001444923100291
A7223).
The interpolation of recombinant human Transferrins,iron complexes (rHTR) utilizes recombinant human Transferrins,iron complexes (rHTR) to replenish the final concentration that said fresh clone's substratum rises to 5mg/.This expression, the fresh clone's culture medium supplemented of 40ml have the 20g/ of 10 μ l rHTR to rise stock solution ( (9701-10)).
The interpolation of RHA and recombinant human Transferrins,iron complexes (rHSA+rHTR): utilizing RHA (rHSA) to replenish said fresh clone's substratum to final concentration is the 2g/ liter, and further to be supplemented to final concentration with recombinant human Transferrins,iron complexes (rHTR) be the 5mg/ liter.This expression, the 20g/ that clone's culture medium supplemented that 40ml is fresh has the 50g/ of 1.6ml rHSA to rise stock solution and 10 μ l rHTR rises stock solution (
Figure BPA00001444923100293
(9701-10)).
According to the clone that the description incubation is dull and stereotyped and counting is produced in material and method part.
The result proves (Fig. 3), if separately use rHSA and rHTR, then it does not improve or only improve on a small quantity cloning efficiency.All of a sudden, when with two kinds of protein combination in same substratum the time, cloning efficiency significantly improves to reach and contains 90% of FBS substratum.
Embodiment 4: add or do not add the titration of the recombinant human Transferrins,iron complexes of RHA
The purpose of experiment is whether two kinds of proteic growth-promoting effects are concentration dependents.
The Clone23 cell be through as in material and the described restricted dilution of method part and artificial clone unicellular.For every kind of substratum combination, all spread two 96 orifice plates.Experimentize according to following:
Positive control (FBS): said clone's culture medium supplemented have 10% from
Figure BPA00001444923100294
the qualified foetal calf serum of hot deactivation (FBS) (10100-147).This expression 40ml clone culture medium supplemented has 4ml FBS (100% stock solution).
The interpolation of recombinant human Transferrins,iron complexes (rHTR): the recombinant human Transferrins,iron complexes (rHTR) that utilizes concentration to improve replenishes said fresh clone's substratum.Rises stock solution (
Figure BPA00001444923100295
(9701-10)) through the 20g/ that adds rHTR and to fresh clone's substratum, adjusted the following final concentration of rHTR: 5mg/ liter, 50mg/ liter, 100mg/ liter and 200mg/ liter.
The interpolation of recombinant human Transferrins,iron complexes and RHA (rHTR+rHSA): utilizing RHA (rHSA) to replenish said fresh clone's substratum to final concentration is the 2g/ liter.All that the concentration maintenance of rHSA is constant in all this experiments.Further be supplemented with the substratum of rHSA with the recombinant human Transferrins,iron complexes (rHTR) of various amounts.Rise in stock solution (
Figure BPA00001444923100301
9701-10) to fresh clone's substratum through the 20g/ that adds rHTR, adjusted the following final concentration of rHTR: 2g/ rises rHSA+5mg/ and rises rHTR; 2g/ rises rHSA+50mg/ and rises rHTR; 2g/ rises rHSA+100mg/ and rises rHTR; 2g/ rises rHSA+200mg/ and rises rHTR.
According to the clone that the description incubation is dull and stereotyped and counting is produced in material and method part.
The result clearly proves (Fig. 4) two kinds of proteic synergies.RHTR can not promote the cell growth separately.Show that in test before only rHSA promotes the cell growth to reach 20% of contrast approximately.Can be observed best promoting growth of cell effect through two kinds of proteic combinations.What is interesting is that very the rHTR of lower concentration (5mg/ liter) is enough to realize that the cell growth reaches 90% of control cultures.
Embodiment 5: utilizing different Chinese hamster ovary celIs through the FACS that is equipped with the automated cell sedimentation unit is the single cell clone experiment of carrying out
The purpose of experiment is test RHA (rHSA) and recombinant human Transferrins,iron complexes (rHTR) and different Chinese hamster ovary celI system combinations.Further purpose be estimate when cell when depositing through the unicellular of FACS clone and through automation cell, this culture medium prescription these cells that whether successfully increased.
CHO Clone23, CHO K1 and CHO DG44 cell are unicellular through material and the described FACS automatically cloning of method part.For every kind of substratum combination, all spread two 96 orifice plates.Experimentize according to following:
Positive control (FBS): said clone's culture medium supplemented have 10% from
Figure BPA00001444923100302
heat-inactivated qualified foetal calf serum (FBS) (10100-147).This expression 40ml clone culture medium supplemented has 4ml FBS (100% stock solution).
The interpolation of RHA and recombinant human Transferrins,iron complexes (rHSA+rHTR): utilizing RHA (rHSA) to replenish said fresh clone's substratum to final concentration is the 2g/ liter, and further to be supplemented to final concentration with recombinant human Transferrins,iron complexes (rHTR) be the 5mg/ liter.This expression, the 20g/ that clone's culture medium supplemented that 40ml is fresh has the 50g/ of 1.6ml rHSA to rise stock solution and 10 μ l rHTR rises stock solution (
Figure BPA00001444923100303
(9701-10)).
According to the clone that the description incubation is dull and stereotyped and counting is produced in material and method part.
The result proves that the combination of rHSA and rHTR is added on the effect (Fig. 5) that promotes through in the sedimentary single celled cell growth of FACS.In addition, these results have shown the validity that this culture medium prescription to different Chinese hamster ovary celIs is.Because the strong growth of CHO K1 cell, when comparing with CHO clone23 or CHODG44 cell, the effect of rHSA and rHTR is not remarkable.Significantly, when when the FACS inoculating cell, the cell that the growth even be superior to of cell in the substratum that is supplemented with rHSA and rHTR grown through restricted dilution artificial inoculation.

Claims (22)

1. a culturing cell crowd in serum-free cell culture medium method, the cell concn of wherein said cell mass is less than 100 cell/ml, and said method comprises the steps:
A) in first serum-free cell culture medium with cell concn culturing cell crowd greater than 100 cell/ml;
B) said cell concn is reduced to less than 100 cell/ml; With
C) in second serum-free cell culture medium, cultivate said cell, wherein said second serum-free cell culture medium comprises recombinant albumin and recombinant transferrin.
2. in serum-free cell culture medium, cultivate single celled method for one kind, said method comprises the steps:
A) in first serum-free cell culture medium culturing cell concentration greater than the cell mass of 100 cell/ml;
B) isolate unicellular from said cell mass; With
C) cultivation is said unicellular in second serum-free cell culture medium, and wherein said second cell culture medium comprises recombinant albumin and recombinant transferrin.
3. as the described method of aforementioned each claim, wherein, when when cultivating the cell of said cell mass greater than the cell concn of 100 cell/ml, said cell does not need recombinant transferrin and/or recombinant albumin to be used for growth.
4. like the described method of aforementioned each claim, wherein, the cell of said cell mass is the cell that can in the substratum that does not contain animal component, grow.
5. like the described method of aforementioned each claim, wherein, the cell of said cell mass is to adapt to the cell of in the substratum that does not contain animal component, growing.
6. like claim 4 or 5 described methods, wherein, the said substratum that does not contain animal component is not protein-contg substratum.
7. like the described method of aforementioned each claim, wherein, recombinant albumin that the said cell culture medium that uses in the step a) comprises and recombinant transferrin lack than the said cell culture medium that uses in the step c).
8. like the described method of aforementioned each claim, wherein, the said cell culture medium that uses in step a) and/or the step c) is the substratum that does not contain animal component.
9. like the described method of aforementioned each claim, said method also comprises the steps:
D) in the 3rd serum-free cell culture medium, cultivate said cell, the said cell culture medium of the concentration ratio step c) of the recombinant transferrin of said the 3rd serum-free cell culture medium and/or recombinant albumin is low.
10. method as claimed in claim 9, wherein, the said cell culture medium of step d) does not contain recombinant transferrin and recombinant albumin.
11. like the described method of aforementioned each claim, the Osmolality of the said cell culture medium of step c) is 280-320mOsmol/kg H 2O, and pH is 6.8-7.1.
12. as the described method of aforementioned each claim, wherein, step a) and c) the said cell culture medium that uses comprises the L-glutaminate of concentration less than 4mM.
13. as the described method of aforementioned each claim, wherein, step a) and c) in the said cell culture medium that uses comprise non-Tf-Fe.
14., wherein, in step c), cultivated said cell at least 6 days like the described method of aforementioned each claim.
15. like the described method of aforementioned each claim, wherein, the said cell of incubation is at least 6 days in step c).
16. as the described method of aforementioned each claim, wherein, volume of culture is 1ml at the most in the step c).
17. as the described method of aforementioned each claim; Wherein, The said cell culture medium that uses in the step c) comprise at least 100mg/ rise recombinant albumin and at least 0.5mg/ rise recombinant transferrin, preferably at least 2000mg/ rise recombinant albumin and at least 5mg/ rise recombinant transferrin.
18., wherein, in step b), separate said unicellular through the automated cell separation system like each described method among the claim 2-17.
19. like the described method of aforementioned each claim, wherein, said cell mass is a Chinese hamster ovary celI system.
Be used for the purposes of culturing cell concentration 20. contain the serum-free cell culture medium of recombinant albumin and recombinant transferrin less than the cell mass of 100 cell/ml.
21. cultured cells concentration is less than the cell mass of 100 cell/ml in the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin.
22. cultured cells concentration is less than the cell mass of 100 cell/ml and expanded cells thus in the serum-free cell culture medium that contains recombinant albumin and recombinant transferrin.
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